Organic chemicals such as organic acids, esters, and polyols can be used to synthesize plastic materials and other products. To meet the increasing demand for organic chemicals, more efficient and cost-effective production methods are being developed which utilize raw materials based on carbohydrates rather than hydrocarbons. For example, certain bacteria have been used to produce large quantities of lactic acid used in the production of polylactic acid. 3-hydroxypropionic acid (3-HP) is an organic acid. Several chemical synthesis routes have been described to produce 3-HP, and biocatalytic routes have also been disclosed (WO 01/116346). 3-HP has utility for specialty synthesis and can be converted to commercially important intermediates by known methods in the chemical industry, e.g., acrylic acid by dehydration, malonic acid by oxidation, esters by esterification reactions with alcohols, and 1,3-propanediol by reduction. The 3-HP compound can be produced biocatalytically from phosphoenolpyruvate or pyruvate through a key beta-alanine (β-alanine) intermediate (FIG. 1). β-alanine can be synthesized in cells from carnosine, β-alanyl arginine, β-alanyl lysine, uracil via 5,6-dihydrouracil and N-carbamoyl-β-alanine, N-acetyl-β-alanine, anserine, or aspartate (FIG. 2). However, these routes may not be cost-effective because they require rare precursors or starting compounds that are more costly than 3-HP. Therefore, production of 3-HP using biocatalytic routes would be more efficient if alpha alanine (α-alanine) could be efficiently converted to β-alanine directly (FIG. 1). A naturally occurring enzyme that inter-converts α-alanine to β-alanine has not yet been identified. It would be advantageous if enzymatic activities that carry out the efficient conversion of α-alanine to β-alanine were identified, such as an alanine 2,3-aminomutase (AAM). Enzymes having very low levels of AAM have been evolved in the laboratory (WO 03/1062173).
There are naturally occurring enzymes known to interconvert lysine to β-lysine. Lysine 2,3-aminomutase (KAM) was first described by Barker in Clostridium SB4 (now C. subterminale) as catalyzing the first step in the fermentation of lysine. KAM has been purified from C. subterminale, the corresponding gene cloned and expressed in E. coli (e.g., U.S. Pat. No. 6,248,874, incorporated herein by reference). The specific activity of purified KAM from C. subterminale SB4 cells has been reported as 30-40 units/mg (Lieder et. al., 1998, Biochemistry 37:2578), where a unit is defined as μmoles lysine/min. The corresponding recombinant KAM had equivalent enzyme activity (34.5±1.6 μmoles lysine/min/mg protein). See U.S. Patent Application Publication No. 2003/0113882, the whole of which is incorporated herein by reference.
Based upon the sequence of the KAM from C. subterminale, KAM genes have been annotated in the genomes of other organisms. However, in most cases, the enzymatic activities of the polypeptides encoded by these genes have not been confirmed. Exceptions are the B. subtilis gene (Chen et al., 2000, Biochem. J. 348:539-549), and the Porphyromonas gingivalis and F. nucleatum genes. The B. subtilis KAM, encoded by the yodO gene, is more resistant to O2 than the C. subterminale KAM, but it is markedly less active. As reported in U.S. Patent Application Publication No. 200310113882, the B. subtilis KAM has a specific KAM activity of only 0.62 U/mg.
C. subterminale SB4 KAM has been reported to have some cross-reactivity with L -alanine, converting it into β-alanine. See U.S. Patent Application Publication No. 2003/0113882 A1. The publications WO 03/062173 and WO 02/42418 disclose the first reports of AAM activity based upon modification of kam genes. In these applications, the synthetic aam genes had AAM activity as detected by the complementation of an E. coli ΔpanD strain. However, because alanine is not the natural substrate for this enzyme, the activity for this conversion is substantially less than the activity for conversion of lysine, its natural substrate. Such low AAM activity would not provide a cost-effective route to converting α-alanine to β-alanine.
The polynucleotide for the wild-type lysine aminomutase (KAM) of Porphyromonas gingivalis, designated as Pgaam, has the polynucleotide sequence of SEQ ID NO:1 (GenBank Accession No. AE017175) and encodes the 416 amino acid residue polypeptide of SEQ ID NO:2. The polynucleotide (SEQ ID NO: 1) encoding this molecule was modified (SEQ ID NO: 3) as described in WO 03/106217, to produce a polypeptide (SEQ ID NO:4), designated as Pgaam2, having a detectible AAM activity. The polypeptide of SEQ ID NO:4, which exhibits a detectible AAM activity, differs from wild-type P. gingivalis Pgaam by having the following seven (7) amino acid substitutions: N19Y, E30K, L53P, H85Q, I192V, D331G, and M342T.
The percent homology between the amino acid sequences of the various KAMs from B. subtilis, C. stricklandii, F. nucleatum, and P. gingivalis are shown in Table 1 below.
TABLE 1B subtilisC. stricklandiiF. nucleatumP. gingivalisB subtilis100%53%50%52%C. stricklandii53%100%69%72%F. nucleatum50%69%100%70%P. gingivalis52%72%70%100%
The present disclosure is directed to engineered alanine 2,3-aminomutases having improved activity over those AAM enzymes known in the art.